PtdIns(3)P accumulation in triple lipid-phosphatase-deletion mutants triggers lethal hyperactivation of the Rho1p/Pkc1p cell-integrity MAP kinase pathway

In the budding yeast Saccharomyces cerevisiae, the regulation of phosphatidylinositol 3-phosphate [PtdIns(3)P] is an essential function shared by the myotubularin-related phosphatase Ymr1p and the synaptojanin-like phosphatases Sjl2p and Sjl3p. The aim of this study was to gain further insight into the mechanisms underlying the toxicity of PtdIns(3)P accumulation in ymr1[Delta] sjl2[Delta] sjl3[Delta] mutant cells. We conducted a genetic screen to isolate genes that, when overexpressed, would rescue the conditional lethality of ymr1[Delta] sjl2[Delta] sjl3[Delta] triple-mutant cells expressing YMR1 from the dextrose-repressible GAL1 promoter. This approach identified 17 genes that promoted growth of the triple mutant on media containing dextrose. Interestingly, the most frequently isolated gene product was a truncated form of PKC1 (Pkc1-T615) that lacked the C-terminal kinase domain. This Pkc1-T615 fragment also rescued the lethality of ymr1[superscript ts] sjl2[Delta] sjl3[Delta] cells at restrictive temperature, and further mapping of the rescuing activity showed that the N-terminal Rho1-GTP-interacting HR1 domains (Pkc1-T242) were sufficient. This indicated that the PKC1 fragments might act by interfering with Rho1-GTP signal propagation. Consistent with this, deletion of the ROM2 gene, which encodes a major Rho1p guanine-nucleotide exchange factor, bypassed the lethal effect of PtdIns(3)P accumulation in ymr1[Delta] sjl2[Delta] sjl3[Delta] triple-mutant cells. Furthermore, cells deficient in phosphoinositide 3-phosphatase (PI 3-phosphatase) activity were defective for Rho1p/Pkc1p pathway regulation, which included an inability of these cells to adapt to heat stress. Taken together, the results of this study indicated that aberrant Rho1p/Pkc1p signaling contributes to the lethal effects of PtdIns(3)P accumulation in cells deficient in PI 3-phosphatase activity.